nf-core/marsseq

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Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

• Download and build references - Build references needer to run the pipeline
• Prepare pipeline
• Label reads
• Align reads
• Demultiplexing
• Quality control
  • QC - Internal QC report
  • FastQC - Raw read QC
  • MultiQC - Aggregate report describing results and QC from the whole pipeline
• Workflow reporting and genomes
  • Pipeline information - Report metrics generated during the workflow execution

The pipeline is executed per Batch and therefore the folder structure looks like following:

results/
|-- multiqc
|-- pipeline_info
|-- references
`-- <batch>

Download and build references

.
└── <genome>
    ├── bowtie2
    │   ├── <genome>.1.bt2
    │   ├── <genome>.2.bt2
    │   ├── <genome>.3.bt2
    │   ├── <genome>.4.bt2
    │   ├── <genome>.rev.1.bt2
    │   └── <genome>.rev.2.bt2
    ├── <genome>.fa
    ├── <genome>.gtf
    ├── star
    │   ├── chrLength.txt
    │   ├── chrNameLength.txt
    │   ├── chrName.txt
    │   ├── chrStart.txt
    │   ├── exonGeTrInfo.tab
    │   ├── exonInfo.tab
    │   ├── geneInfo.tab
    │   ├── Genome
    │   ├── genomeParameters.txt
    │   ├── Log.out
    │   ├── SA
    │   ├── SAindex
    │   ├── sjdbInfo.txt
    │   ├── sjdbList.fromGTF.out.tab
    │   ├── sjdbList.out.tab
    │   └── transcriptInfo.tab
    └── versions.yml

The pipeline downloads references from GENCODE database. This is required, because the MARS-seq is using ERCC spike-ins, which have to be appended. Next it builds bowtie2 index. If --velocity flag is set, star index is also built.

Prepare pipeline

At the beginning the pipeline validates the input *xlsx files which are further converted into .txt files. The majority of internal tools are written in Perl by the original authors. The nf-core/marsseq is trying to simplify these steps while replicating the necessary formats and executions in the background. Because the pipeline uses bowtie2 as a default aligner, reads are furher split into subfiles of 4,000,000 reads per file so the aligner doesn’t run out of memory.

Label reads

In this intermediate step, reads are further processed and barcodes (CB + UMI) is transferred from R2 to R1. This is important as the CB is further used in the demultiplexing step.

• R1 structure: LA (3bp) + PB (4bp) + cDNA (66bp) + RA (2bp)
• R2 structure: CB (7bp) + UMI (8bp)

where

• LA: left adapter
• PB: pool barcode
• RA: right adapter
• CB: cell barcode
• UMI: Unique Molecular Indentifier

In this step, files are not saved in the overal results, they reside in the work folder.

Align reads

Split reads are aligned using bowtie2. Next, all the aligned reads are merged into one SAM file which is used as an input for demultiplexing.

If --velocity flag is set, the reads are also aligned using StarSolo to estimated both spliced and unspliced reads which can be used for RNA velocity estimation. This is an additional plugin which we developed. In short MARS-seq2.0 reads are converted to 10X v2 format. Additionally, a whitelist is generated for aligned to perform demultiplexing.

Demultiplexing

Demultiplexing step where pipeline generates a count matrix for invidivual amplification batch. The count matrix can be found in <batch>/output/umi.tab. The information above have been taken from original documentation for MARS-seq2.0 which can be round here.

QC

QC report

Internal script for generating QC report after bowtie2 alignment and demultiplexing.

FastQC

FastQC gives general quality metrics about your sequenced reads. It provides information about the quality score distribution across your reads, per base sequence content (%A/T/G/C), adapter contamination and overrepresented sequences. For further reading and documentation see the FastQC help pages.

NB: The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequence and potentially regions with low quality.

MultiQC

MultiQC is a visualization tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in the report data directory.

Results generated by MultiQC collate pipeline QC from supported tools e.g. FastQC. The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability. For more information about how to use MultiQC reports, see http://multiqc.info.

Workflow reporting and genomes

Pipeline information

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.